 Plants utilize the sun energy in a process called photosynthesis.
 The chloroplasts in the plant leaves harness that light and convert it into
chemical energy.
 Plants are called autotrophs because they use sunlight energy directly and
combine inorganic compounds to form organic molecules.
 Heterotrophs, on the other hand, obtain their energy by consuming other
life forms. Humans are an example of heterotrophs.
Balanced equation for photosynthesis
6 CO2
carbon
dioxide
+ 12 H2O + light energy
water
(sun)
C6H12O6 + 6 O2 + 6 H2O
glucose
oxygen
water
2
wavelengths
3
 http://www.youtube.com/watch?feature=player_embe
dded&v=MkUGz6kEQMg
4
 A spectrophotometer can be used
to measure absorption and
transmittance of light through
solutions.
 Go back to the chapter of
spectrophotometry to understand
the use of the blank (B)
C B
S
 Experiment
 Carotene (C) and spinach (S)
extracts will be tested regarding
their absorption and transmittance
at different wavelengths.
5
WL= wavelength
%T = transmittance
Carrot extract
WL
%T
WL
%T
6
Spinach extract
WL
%T
WL
%T
7
Transmittance (%)
Wave length (nm)
If you invert this graph, you will see the classic absorbance graph found in your text book.
8
Absorbance
Inverted graph
Carotene
Spinach
400
500
625
675
WL (nm)
9
 The same procedure was done, but reading the absorbance of both
spinach and carrot extracts.
 The data was used to draw the graph shown in the next slide.
WL
400 425
450
475
500
525 550
575 600 625
650 675
700
S
0.35 0.5
0.7
0.8
0.6
0.2
0.1
0.2 0.5
0.35
0.3
0.2
0.1
C
0.8
0.8
0.75 0.7
0.6 0.6
0.5 0.1
0.1
0.1
0.1
0.2
0.8
10
absorbance
Wavelength (nm)
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 Is used to separate substances from one
another based on their characteristics.
 This exercise will separate pigments of a
spinach leaf.
 The separation is due to the solubility of the
pigment in the chromatography solvent
(10% acetone in petroleum ether) and the
affinity of the pigment to the paper surface.
solvent
Roll of chromatography paper
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Adding pigment to the paper
Paper strip
 On a 3 cm x 20 cm strip of
chromatography paper, a baseline is
drawn with pencil (graphite) about 2 cm
from the bottom. On this pencil line the
spinach leaf pigment is deposited by
rolling a coin over the leaf, onto the paper.
Graphite line
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 Place the strip into the chamber.
 The paper with pigment is placed into a
test tube containing 1 cm of
chromatography solvent.
Solvent
movement
 Left undisturbed for 10-15 minutes the
solvent moves up the paper and carries
with it the pigments of the spinach leaf.
The pigments move at their own
characteristic rate.
solvent
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 There are four pigments that are seen on the chromatogram.
 Carotene (orange)
 Xanthophyll (yellow)
 Chlorophyll a (blue-green)
 Chlorophyll b (yellow-green)
 Which pigment has the greatest
carotene
affinity for the solvent?
 Which has the greatest affinity for the
paper?
Chlorophyll b
Baseline
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 Determine the Rf (ratio factor)
Solvent line
value of each pigment.
 To determine the Rf value of a pigment you
must measure the distance traveled by the
pigment/distance traveled by the solvent.
 To determine distance traveled by the
pigment you must measure from the
middle of the pigment band to the
baseline.
 To determine the distance traveled by the
solvent you must measure from the solvent
line to the baseline.
baseline
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 This experiment proves that the most common form of carbohydrate stored by
plants is starch, a polymer of glucose.
 To test for starch, the leaf pigments must first be removed by placing it in boiling
95% ethanol.
Coleus sp.
Ethanol cannot be boiled directly on the
hotplate. A beaker containing ethanol is placed
into a larger beaker of boiling water
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 Iodine is then added to the leaf. If starch is present the area will
become blue-black .
Before boiling
The End
After boiling and staining
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